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1.  A Developmental Framework for Graft Formation and Vascular Reconnection in Arabidopsis thaliana 
Current biology : CB  2015;25(10):1306-1318.
Plant grafting is a biologically important phenomenon involving the physical joining of two plants to generate a chimeric organism. It is widely practiced in horticulture and used in science to study the long distance movement of molecules. Despite its widespread use, the mechanism of graft formation and vascular reconnection is not well understood. Here, we study the dynamics and mechanisms of vascular regeneration in Arabidopsis thaliana during graft formation when the vascular strands are severed and reconnected.
We demonstrate a temporal separation between tissue attachment, phloem connection, root growth and xylem connection. By analysing cell division patterns and hormone responses at the graft junction, we found that tissues initially show an asymmetry in cell division, cell differentiation and gene expression, and through contact with the opposing tissue, lose this asymmetry and reform the vascular connection. In addition, we identified genes involved in vascular reconnection at the graft junction, and demonstrate that these auxin response genes are required below the graft junction.
We propose an inter-tissue communication process that occurs at the graft junction and promotes vascular connection by tissue-specific auxin responses involving ABERRANT LATERAL ROOT FORMATION 4 (ALF4). Our study has implications for phenomena where forming vascular connections are important including graft formation, parasitic plant infection and wound healing.
PMCID: PMC4798781  PMID: 25891401
2.  Connective Auxin Transport in the Shoot Facilitates Communication between Shoot Apices 
PLoS Biology  2016;14(4):e1002446.
The bulk polar movement of the plant signaling molecule auxin through the stem is a long-recognized but poorly understood phenomenon. Here we show that the highly polar, high conductance polar auxin transport stream (PATS) is only part of a multimodal auxin transport network in the stem. The dynamics of auxin movement through stems are inconsistent with a single polar transport regime and instead suggest widespread low conductance, less polar auxin transport in the stem, which we term connective auxin transport (CAT). The bidirectional movement of auxin between the PATS and the surrounding tissues, mediated by CAT, can explain the complex auxin transport kinetics we observe. We show that the auxin efflux carriers PIN3, PIN4, and PIN7 are major contributors to this auxin transport connectivity and that their activity is important for communication between shoot apices in the regulation of shoot branching. We propose that the PATS provides a long-range, consolidated stream of information throughout the plant, while CAT acts locally, allowing tissues to modulate and be modulated by information in the PATS.
The growing shoot tips of a plant can communicate and influence each other’s growth. This study reveals that this communication is mediated by a multimodal auxin transport network in the stem, with complex dynamics.
Author Summary
The growing shoot tips of a plant can communicate and influence each other’s growth. This is most obvious in the phenomenon of apical dominance, in which the leading shoot in a shoot system inhibits the growth of buds in the axils of the leaves below it. Removing the leading shoot results in the activation of these buds to produce branches. It has been known for almost a century that the plant hormone auxin plays a central part in apical dominance. Auxin is produced in growing shoot tips and transported down the main stem in the Polar Auxin Transport Stream (PATS). We have previously shown that communication between shoot tips is mediated at least in part by competition between buds for access to the PATS. Here we show that this communication depends not only on the highly polar, high-capacity transport of the PATS, which is localized to the vascular bundles of the stem, but also on widespread less polar transport activities, which act to exchange information between the PATS and surrounding tissues. We term this Connective Auxin Transport (CAT) and show that the exchange of auxin between CAT and PATS is important for bud-bud communication.
PMCID: PMC4847802  PMID: 27119525
3.  Cytokinin is required for escape but not release from auxin mediated apical dominance 
The Plant Journal  2015;82(5):874-886.
Auxin produced by an active primary shoot apex is transported down the main stem and inhibits the growth of the axillary buds below it, contributing to apical dominance. Here we use Arabidopsis thaliana cytokinin (CK) biosynthetic and signalling mutants to probe the role of CK in this process. It is well established that bud outgrowth is promoted by CK, and that CK synthesis is inhibited by auxin, leading to the hypothesis that release from apical dominance relies on an increased supply of CK to buds. Our data confirm that decapitation induces the expression of at least one ISOPENTENYLTRANSFERASE (IPT) CK biosynthetic gene in the stem. We further show that transcript abundance of a clade of the CK-responsive type-A Arabidopsis response regulator (ARR) genes increases in buds following CK supply, and that, contrary to their typical action as inhibitors of CK signalling, these genes are required for CK-mediated bud activation. However, analysis of the relevant arr and ipt multiple mutants demonstrates that defects in bud CK response do not affect auxin-mediated bud inhibition, and increased IPT transcript levels are not needed for bud release following decapitation. Instead, our data suggest that CK acts to overcome auxin-mediated bud inhibition, allowing buds to escape apical dominance under favourable conditions, such as high nitrate availability.
Significance Statement
It has been proposed that the release of buds from auxin-mediated apical dominance following decapitation requires increased cytokinin biosynthesis and consequent increases in cytokinin supply to buds. Here we show that in Arabidopsis, increases in cytokinin appear to be unnecessary for the release of buds from apical dominance, but rather allow buds to escape the inhibitory effect of apical auxin, thereby promoting bud activation in favourable growth conditions.
PMCID: PMC4691322  PMID: 25904120
cytokinin; auxin; shoot branching; apical dominance; Arabidopsis thaliana; Isopentenyltransferase; type-A Arabidopsis response regulators
5.  The Tinkerbell (Tink) Mutation Identifies the Dual-Specificity MAPK Phosphatase INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5) as a Novel Regulator of Organ Size in Arabidopsis 
PLoS ONE  2015;10(7):e0131103.
Mitogen-activated dual-specificity MAPK phosphatases are important negative regulators in the MAPK signalling pathways responsible for many essential processes in plants. In a screen for mutants with reduced organ size we have identified a mutation in the active site of the dual-specificity MAPK phosphatase INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5) that we named tinkerbell (tink) due to its small size. Analysis of the tink mutant indicates that IBR5 acts as a novel regulator of organ size that changes the rate of growth in petals and leaves. Organ size and shape regulation by IBR5 acts independently of the KLU growth-regulatory pathway. Microarray analysis of tink/ibr5-6 mutants identified a likely role for this phosphatase in male gametophyte development. We show that IBR5 may influence the size and shape of petals through auxin and TCP growth regulatory pathways.
PMCID: PMC4492785  PMID: 26147117
6.  Three ancient hormonal cues co-ordinate shoot branching in a moss 
eLife  null;4:e06808.
Shoot branching is a primary contributor to plant architecture, evolving independently in flowering plant sporophytes and moss gametophytes. Mechanistic understanding of branching is largely limited to flowering plants such as Arabidopsis, which have a recent evolutionary origin. We show that in gametophytic shoots of Physcomitrella, lateral branches arise by re-specification of epidermal cells into branch initials. A simple model co-ordinating the activity of leafy shoot tips can account for branching patterns, and three known and ancient hormonal regulators of sporophytic branching interact to generate the branching pattern- auxin, cytokinin and strigolactone. The mode of auxin transport required in branch patterning is a key divergence point from known sporophytic pathways. Although PIN-mediated basipetal auxin transport regulates branching patterns in flowering plants, this is not so in Physcomitrella, where bi-directional transport is required to generate realistic branching patterns. Experiments with callose synthesis inhibitors suggest plasmodesmal connectivity as a potential mechanism for transport.
eLife digest
Most land plants have shoots that form branches and plants can regulate when and where they grow these branches to best exploit their environment. Plants with flowers and the more ancient mosses both have branching shoots, but these two groups of plants evolved to grow in this way independently of each other. Most studies on shoot branching have focused on flowering plants and so it is less clear how branching works in mosses.
Three plant hormones—called auxin, cytokinin and strigolactone—control shoot branching in flowering plants. Auxin moves down the main shoot of the plant to prevent new branches from forming. This movement is controlled by the PIN proteins and several other families of proteins. On the other hand, cytokinin promotes the growth of new branches; and strigolactone can either promote or inhibit shoot branching depending on how the auxin is travelling around the plant.
Coudert, Palubicki et al. studied shoot branching in a species of moss called Physcomitrella patens. The experiments show that cells on the outer surface of the main shoot are essentially reprogrammed to become so-called ‘branch initials’, which will then develop into new branches. Next, Coudert, Palubicki et al. made a computational model that was able to simulate the pattern of shoot branching in the moss.
Further experiments supported the predictions made by the model. Coudert, Palubicki et al. found that, as in flowering plants, auxin from the tip of the main shoot suppresses branching in the moss, and cytokinin promotes branching. The experiments also showed that strigolactone inhibits shoot branching, but its role is restricted to the base of the shoots. The model predicts that, unlike in flowering plants, auxin must flow in both directions in moss shoots to produce the observed patterns of shoot branching. Also, the experiments suggest that the PIN proteins and another group of proteins that control the movement of auxin do not regulate shoot branching in moss. Instead, it appears that auxin may move through microscopic channels that link one moss cell to the next.
Coudert, Palubicki et al.'s findings suggest that both flowering plants and mosses have evolved to use the same three hormones to control shoot branching, but that these hormones interact in different ways. One key next step will be to find out how auxin is transported during shoot branching in moss by manipulating the opening of the channels between the cells. A further challenge will be to find out the precise details of how the hormones control the activity of the branch initial cells.
PMCID: PMC4391503  PMID: 25806686
Physcomitrella; branching; apical dominance; gametophyte; other
7.  The culture of scientific research 
F1000Research  2015;4:66.
In 2014, the UK-based Nuffield Council on Bioethics carried out a series of engagement activities, including an online survey to which 970 people responded, and 15 discussion events at universities around the UK to explore the culture of research in the UK and its effect on ethical conduct in science and the quality of research. The findings of the project were published in December 2014 and the main points are summarised here. We found that scientists are motivated in their work to find out more about the world and to benefit society, and that they believe collaboration, multidisciplinarity, openness and creativity are important for the production of high quality science. However, in some cases, our findings suggest, the culture of research in higher education institutions does not support or encourage these goals or activities. For example, high levels of competition and perceptions about how scientists are assessed for jobs and funding are reportedly contributing to a loss of creativity in science, less collaboration and poor research practices. The project led to suggestions for action for funding bodies, research institutions, publishers and editors, professional bodies and individual researchers.
PMCID: PMC4376168  PMID: 25866623
Research; culture; science; integrity; misconduct; ethics; Nuffield Council on Bioethics
8.  Functional screening of willow alleles in Arabidopsis combined with QTL mapping in willow (Salix) identifies SxMAX4 as a coppicing response gene 
Plant Biotechnology Journal  2014;12(4):480-491.
Willows (Salix spp.) are important biomass crops due to their ability to grow rapidly with low fertilizer inputs and ease of cultivation in short-rotation coppice cycles. They are relatively undomesticated and highly diverse, but functional testing to identify useful allelic variation is time-consuming in trees and transformation is not yet possible in willow. Arabidopsis is heralded as a model plant from which knowledge can be transferred to advance the improvement of less tractable species. Here, knowledge and methodologies from Arabidopsis were successfully used to identify a gene influencing stem number in coppiced willows, a complex trait of key biological and industrial relevance. The strigolactone-related More AXillary growth (MAX) genes were considered candidates due to their role in shoot branching. We previously demonstrated that willow and Arabidopsis show similar response to strigolactone and that transformation rescue of Arabidopsis max mutants with willow genes could be used to detect allelic differences. Here, this approach was used to screen 45 SxMAX1, SxMAX2, SxMAX3 and SxMAX4 alleles cloned from 15 parents of 11 mapping populations varying in shoot-branching traits. Single-nucleotide polymorphism (SNP) frequencies were locus dependent, ranging from 29.2 to 74.3 polymorphic sites per kb. SxMAX alleles were 98%–99% conserved at the amino acid level, but different protein products varying in their ability to rescue Arabidopsis max mutants were identified. One poor rescuing allele, SxMAX4D, segregated in a willow mapping population where its presence was associated with increased shoot resprouting after coppicing and colocated with a QTL for this trait.
PMCID: PMC4238783  PMID: 24393130
Arabidopsis; allelic diversity; mutant rescue; Salix; QTL; strigolactone
9.  Moving beyond the GM Debate 
PLoS Biology  2014;12(6):e1001887.
A secure, environmentally sustainable food supply is agreed to be a vital priority. In this Perspective, Ottoline Leyser explains why she believes that progress toward this important goal is side-tracked by unproductive debates about the role of genetic modification.
Once again, there are calls to reopen the debate on genetically modified (GM) crops. I find these calls frustrating and unnecessarily decisive. In my opinion the GM debate, on both sides, continues to hamper the urgent need to address the diverse and pressing challenges of global food security and environmental sustainability. The destructive power of the debate comes from its conflation of unrelated issues, coupled with deeply rooted misconceptions of the nature of agriculture.
PMCID: PMC4051613  PMID: 24914954
10.  Paralogous Radiations of PIN Proteins with Multiple Origins of Noncanonical PIN Structure 
Molecular Biology and Evolution  2014;31(8):2042-2060.
The plant hormone auxin is a conserved regulator of development which has been implicated in the generation of morphological novelty. PIN-FORMED1 (PIN) auxin efflux carriers are central to auxin function by regulating its distribution. PIN family members have divergent structures and cellular localizations, but the origin and evolutionary significance of this variation is unresolved. To characterize PIN family evolution, we have undertaken phylogenetic and structural analyses with a massive increase in taxon sampling over previous studies. Our phylogeny shows that following the divergence of the bryophyte and lycophyte lineages, two deep duplication events gave rise to three distinct lineages of PIN proteins in euphyllophytes. Subsequent independent radiations within each of these lineages were taxonomically asymmetric, giving rise to at least 21 clades of PIN proteins, of which 15 are revealed here for the first time. Although most PIN protein clades share a conserved canonical structure with a modular central loop domain, a small number of noncanonical clades dispersed across the phylogeny have highly divergent protein structure. We propose that PIN proteins underwent sub- and neofunctionalization with substantial modification to protein structure throughout plant evolution. Our results have important implications for plant evolution as they suggest that structurally divergent PIN proteins that arose in paralogous radiations contributed to the convergent evolution of organ systems in different land plant lineages.
PMCID: PMC4104312  PMID: 24758777
auxin; auxin transport; PIN protein; plant evolution; phylogeny; protein structure
11.  Strigolactone Can Promote or Inhibit Shoot Branching by Triggering Rapid Depletion of the Auxin Efflux Protein PIN1 from the Plasma Membrane 
PLoS Biology  2013;11(1):e1001474.
Shoot branching is regulated by competition between branches to export the phytohormone auxin into the main stem. The phytohormone strigolactone balances shoot system growth by making auxin export harder to establish, thus modulating the auxin transport network.
Plants continuously extend their root and shoot systems through the action of meristems at their growing tips. By regulating which meristems are active, plants adjust their body plans to suit local environmental conditions. The transport network of the phytohormone auxin has been proposed to mediate this systemic growth coordination, due to its self-organising, environmentally sensitive properties. In particular, a positive feedback mechanism termed auxin transport canalization, which establishes auxin flow from active shoot meristems (auxin sources) to the roots (auxin sinks), has been proposed to mediate competition between shoot meristems and to balance shoot and root growth. Here we provide strong support for this hypothesis by demonstrating that a second hormone, strigolactone, regulates growth redistribution in the shoot by rapidly modulating auxin transport. A computational model in which strigolactone action is represented as an increase in the rate of removal of the auxin export protein, PIN1, from the plasma membrane can reproduce both the auxin transport and shoot branching phenotypes observed in various mutant combinations and strigolactone treatments, including the counterintuitive ability of strigolactones either to promote or inhibit shoot branching, depending on the auxin transport status of the plant. Consistent with this predicted mode of action, strigolactone signalling was found to trigger PIN1 depletion from the plasma membrane of xylem parenchyma cells in the stem. This effect could be detected within 10 minutes of strigolactone treatment and was independent of protein synthesis but dependent on clathrin-mediated membrane trafficking. Together these results support the hypothesis that growth across the plant shoot system is balanced by competition between shoot apices for a common auxin transport path to the root and that strigolactones regulate shoot branching by modulating this competition.
Author Summary
Plants can adapt their form to suit the environment in which they are growing. For example, genetically identical plants can develop as a single unbranched stem or as a highly ramified bush. This broad developmental potential is possible because the shoot system is produced continuously by growing tips, known as shoot meristems. Meristems produce the stem and leaves of a shoot, and at the base of each leaf, a new meristem is formed. This meristem can remain dormant as a small bud or activate to produce a branch. Thus, the shoot system is a community of shoot meristems, the combined activity and inactivity of which shape shoot form. Here we provide evidence that growth is balanced across the Arabidopsis shoot system by competition between the shoot meristems. This competition is likely mediated by the requirement of meristems to export the plant hormone auxin in order to activate bud outgrowth. In our model, auxin in the main stem, exported from active branches, can prevent auxin export by dormant buds, thus preventing their activation. Our findings show that a second hormone, strigolactone, increases the level of competition between branches by making auxin export harder to establish. Together, these hormones balance growth across the shoot system, adjusting it according to the environmental conditions in which a plant is growing.
PMCID: PMC3558495  PMID: 23382651
12.  Mutation of the cytosolic ribosomal protein-encoding RPS10B gene affects shoot meristematic function in Arabidopsis 
BMC Plant Biology  2012;12:160.
Plant cytosolic ribosomal proteins are encoded by small gene families. Mutants affecting these genes are often viable, but show growth and developmental defects, suggesting incomplete functional redundancy within the families. Dormancy to growth transitions, such as the activation of axillary buds in the shoot, are characterised by co-ordinated upregulation of ribosomal protein genes.
A recessive mutation in RPS10B, one of three Arabidopsis genes encoding the eukaryote-specific cytoplasmic ribosomal protein S10e, was found to suppress the excessive shoot branching mutant max2-1. rps10b-1 mildly affects the formation and separation of shoot lateral organs, including the shoot axillary meristems. Axillary meristem defects are enhanced when rps10b-1 is combined with mutations in REVOLUTA, AUXIN-RESISTANT1, PINOID or another suppressor of max2-1, FAR-RED ELONGATED HYPOCOTYL3. In some of these double mutants, the maintenance of the primary shoot meristem is also affected. In contrast, mutation of ALTERED MERISTEM PROGRAMME1 suppresses the rps10b-1axillary shoot defect. Defects in both axillary shoot formation and organ separation were enhanced by combining rps10b-1 with cuc3, a mutation affecting one of three Arabidopsis NAC transcription factor genes with partially redundant roles in these processes. To assess the effect of rps10b-1 on bud activation independently from bud formation, axillary bud outgrowth on excised cauline nodes was analysed. The outgrowth rate of untreated buds was reduced only slightly by rps10b-1 in both wild-type and max2-1 backgrounds. However, rps10b-1 strongly suppressed the auxin resistant outgrowth of max2-1 buds. A developmental phenotype of rps10b-1, reduced stamen number, was complemented by the cDNA of another family member, RPS10C, under the RPS10B promoter.
RPS10B promotes shoot branching mainly by promoting axillary shoot development. It contributes to organ boundary formation and leaf polarity, and sustains max2-1 bud outgrowth in the presence of auxin. These processes require the auxin response machinery and precise spatial distribution of auxin. The correct dosage of protein(s) involved in auxin-mediated patterning may be RPS10B-dependent. Inability of other RPS10 gene family members to maintain fully S10e levels might cause the rps10b-1 phenotype, as we found no evidence for unique functional specialisation of either RPS10B promoter or RPS10B protein.
PMCID: PMC3492191  PMID: 22963533
Shoot branching suppressor; S10e; Axillary bud; Leaf polarity; Lateral organ boundary; Auxin; Strigolactone; CUC; REV
13.  Auxin, cytokinin and the control of shoot branching 
Annals of Botany  2011;107(7):1203-1212.
It has been known for many decades that auxin inhibits the activation of axillary buds, and hence shoot branching, while cytokinin has the opposite effect. However, the modes of action of these two hormones in branching control is still a matter of debate, and their mechanisms of interaction are equally unresolved.
Here we review the evidence for various hypotheses that have been put forward to explain how auxin and cytokinin influence axillary bud activity. In particular we discuss the roles of auxin and cytokinin in regulating each other's synthesis, the cell cycle, meristem function and auxin transport, each of which could affect branching. These different mechanisms have implications for the main site of hormone action, ranging from systemic action throughout the plant, to local action at the node or in the bud meristem or leaves. The alternative models have specific predictions, and our increasing understanding of the molecular basis for hormone transport and signalling, cell cycle control and meristem biology is providing new tools to enable these predictions to be tested.
PMCID: PMC3091808  PMID: 21504914
Shoot branching; axillary bud; dormancy; auxin; cytokinin; canalization; polar auxin transport stream; cell cycle
14.  Strigolactone regulation of shoot branching in chrysanthemum (Dendranthema grandiflorum) 
Journal of Experimental Botany  2010;61(11):3069-3078.
Previous studies of highly branched mutants in pea (rms1–rms5), Arabidopsis thaliana (max1–max4), petunia (dad1–dad3), and rice (d3, d10, htd1/d17, d14, d27) identified strigolactones or their derivates (SLs), as shoot branching inhibitors. This recent discovery offers the possibility of using SLs to regulate branching commercially, for example, in chrysanthemum, an important cut flower crop. To investigate this option, SL physiology and molecular biology were studied in chrysanthemum (Dendranthema grandiflorum), focusing on the CCD8/MAX4/DAD1/RMS1/D10 gene. Our results suggest that, as has been proposed for Arabidopsis, the ability of SLs to inhibit bud activity depends on the presence of a competing auxin source. The chrysanthemum SL biosynthesis gene, CCD8 was cloned, and found to be regulated in a similar, but not identical way to known CCD8s. Expression analyses revealed that DgCCD8 is predominantly expressed in roots and stems, and is up-regulated by exogenous auxin. Exogenous SL can down-regulate DgCCD8 expression, but this effect can be overridden by apical auxin application. This study provides evidence that SLs are promising candidates to alter the shoot branching habit of chrysanthemum.
PMCID: PMC2892150  PMID: 20478970
Auxin; CCD8; chrysanthemum; shoot branching; strigolactone
15.  pax1-1 partially suppresses gain-of-function mutations in Arabidopsis AXR3/IAA17 
BMC Plant Biology  2007;7:20.
The plant hormone auxin exerts many of its effects on growth and development by controlling transcription of downstream genes. The Arabidopsis gene AXR3/IAA17 encodes a member of the Aux/IAA family of auxin responsive transcriptional repressors. Semi-dominant mutations in AXR3 result in an increased amplitude of auxin responses due to hyperstabilisation of the encoded protein. The aim of this study was to identify novel genes involved in auxin signal transduction by screening for second site mutations that modify the axr3-1 gain-of-function phenotype.
We present the isolation of the partial suppressor of axr3-1 (pax1-1) mutant, which partially suppresses almost every aspect of the axr3-1 phenotype, and that of the weaker axr3-3 allele. axr3-1 protein turnover does not appear to be altered by pax1-1. However, expression of an AXR3::GUS reporter is reduced in a pax1-1 background, suggesting that PAX1 positively regulates AXR3 transcription. The pax1-1 mutation also affects the phenotypes conferred by stabilising mutations in other Aux/IAA proteins; however, the interactions are more complex than with axr3-1.
We propose that PAX1 influences auxin response via its effects on AXR3 expression and that it regulates other Aux/IAAs secondarily.
PMCID: PMC1855327  PMID: 17430601
16.  Root system architecture determines fitness in an Arabidopsis mutant in competition for immobile phosphate ions but not for nitrate ions. 
Plant root systems often have complex branching patterns. Models indicate that a complex architecture is only required for the acquisition of immobile resources, such as phosphate; mobile ions, notably nitrate, can be effectively taken up by very restricted root systems. We have tested this prediction using the axr4 mutation of Arabidopsis thaliana, the principal phenotypic effect of which is to reduce the number of lateral roots. Arabidopsis thaliana is not a host for mycorrhizal fungi and so acquires all its nutrients through the root system. In both a pot experiment and a field experiment conducted under natural conditions for A. thaliana, we found that only phosphate, and not nitrate, affected the fitness of the mutant relative to the isogenic wild-type line, Columbia. These results confirm model predictions and have implications both for the evolution of complex root systems and for the design of efficient root systems for crops.
PMCID: PMC1691122  PMID: 12396500
17.  Functional Genomics at the Arabidopsis Meeting 
Yeast (Chichester, England)  2000;17(3):235-237.
PMCID: PMC2448372  PMID: 11025535

Results 1-17 (17)